DNA polymerases catalyze the replication of cellular DNA and play an integral role in DNA repair. Translesion synthesis, i.e. DNA synthesis across damaged DNA nucleotides, is a critical step in mutagenesis and the development of cancer. More than ten proteins capable of DNA synthesis have been identified in humans. These DNA polymerases differ in their fidelity and ability to perform translesion synthesis. The goal of this project is to solve the three dimensional structure of DNA polymerases from four different families poised to perform translesion synthesis. These structures will allow examination of the general hypothesis that interactions in the DNA polymerase active site modulate the miscoding and/or efficiency of translesion synthesis. The specific aims involve studies of structural and kinetic mechanisms of (a) error-prone translesion synthesis by high fidelity DNA polymerases ( Families A, B and X) (b) Iesion-blocked DNA synthesis and (c) accurate and error-prone synthesis by specialized translesion synthesis polymerases (Family Y). X-ray crystallography will be used to capture DNA polymerases complexed with oxidatively damaged DNA during various steps in this process. Coupled with steady-state kinetic analysis and site-directed mutagenesis, these structures should reveal structural and functional differences between Iow-(translesion) and high-fidelity DNA polymerases. Structural studies with high-fidelity polymerases will provide insight into the preferential incorporation of dCTP or dATP opposite 8-oxo-dG, establish a structural basis for the "A-rule", and explain the miscoding potential of epsilon-dC and other exocyclic DNA adducts. The mechanism by which abasic sites and thymine glycol block the progression of DNA polymerases will also be examined. Structural analysis of two different Y-family (translesion) DNA polymerases, mouse pol Kappa and human pol eta, will provide insight into their substrate specificity. This project will analyze the mechanism of translesion synthesis at the atomic level and will complement studies of miscoding and mutagenesis (Project 1), the determination of thermodynamic parameters (Project 3) and NMR studies on DNA containing site-specifically placed lesions (Project 4). Overall, this project provides critical knowledge relevant to the structural biology of mutagenesis and human cancer.